Abstract

The host cell factors TRIM5alpha(hu) and Fv-1 restrict N-tropic murine leukemia virus (N-MLV) infection at an early postentry step before or after reverse transcription, respectively. Interestingly, the identity of residue 110 of the MLV capsid determines susceptibility to both TRIM5alpha(hu) and Fv-1. In this study, we investigate the fate of the MLV capsid in cells expressing either the TRIM5alpha(hu) or Fv-1 restriction factor. The expression of TRIM5alpha(hu), but not Fv-1, specifically promoted the premature conversion of particulate N-MLV capsids within infected cells to soluble capsid proteins. The TRIM5alpha(hu)-mediated disassembly of particulate N-MLV capsids was dependent upon residue 110 of the viral capsid. Furthermore, the deletion or disruption of TRIM5alpha(hu) domains necessary for potent N-MLV restriction completely abrogated the disappearance of particulate N-MLV capsids observed with wild-type TRIM5alpha(hu). These results suggest that premature disassembly of the viral capsid contributes to the restriction of N-MLV infection by TRIM5alpha(hu), but not by Fv-1.

Assay to follow the fate of the MLV capsid within infected cells. (A) MDTF cells transduced with an empty LPCX control vector were infected with either VSV G-pseudotyped (Env+) N-MLV or envelope-deficient (Env−) N-MLV virus-like particles for 4 h. Cell lysates were then prepared, and an aliquot was used for Western blotting with an antibody directed against the MLV p30 capsid protein (left). The remaining cell lysates were analyzed on gradients formed with the indicated concentrations of sucrose (weight per volume). The pellets were Western blotted using an antibody directed against the MLV p30 capsid protein. (B) MDTF cells transduced with an empty LPCX control vector were infected with either VSV G-pseudotyped (Env+) or envelope-deficient (Env−) N-MLV or B-MLV virus-like particles for 4 h. Cell lysates were then prepared and analyzed on gradients formed with the indicated concentrations of sucrose (weight per volume). The total input and pellets were Western blotted using an antibody directed against the p30 capsid protein. (C) MDTF cells stably transduced with either an empty LPCX control vector or an LPCX vector expressing TRIM5αhu were infected with either VSV G-pseudotyped (Env+) or envelope-deficient (Env−) N-MLV-GFP for 2 h and analyzed using the fate-of-capsid assay, as described in Materials and Methods. The total input, supernatants, and pellets were Western blotted using an antibody directed against the p30 capsid protein. (D) MDTF cells transduced with either an empty LPCX control vector or an LPCX vector expressing TRIM5αhu were infected with either VSV G-pseudotyped (Env+) or envelope-deficient (Env−) N-MLV-GFP for 2 h and analyzed by the fate-of-capsid assay. The presence of viral RNA within the pelleted cores was detected by RT-PCR, as described in Materials and Methods. The amount of viral RNA in the pellet associated with the LPCX cells exposed to (Env+) N-MLV-GFP was standardized to 100% and set as a reference for comparison to the RNA levels associated with the LPCX-transduced cells exposed to (Env−) N-MLV-GFP and the TRIM5αhu-expressing cells exposed to the (Env+) N-MLV-GFP. The error bars indicate standard deviations.

Effect of the expression of a restricting TRIMα protein on the amounts of particulate N-MLV capsids in the cytosol of infected cells. (A) Steady-state expression levels of the TRIM proteins are shown. Lysates from MDTF or NIH 3T3 cells transduced with either an empty LPCX vector or LPCX vectors expressing HA-tagged TRIM proteins were subjected to Western blotting using an anti-HA antibody. The lysates were also blotted against β-actin to control for total protein. “Mouse” refers to the mouse 9230105E10 protein, used here as a negative control. (B) MDTF cells transduced with LPCX vectors expressing the indicated TRIM proteins were infected with various doses of N-MLV-GFP or B-MLV-GFP. GFP-positive cells were quantified by FACS analysis. (C) MDTF or NIH 3T3 cells stably transduced with either an empty LPCX control vector or an LPCX vector expressing the indicated TRIM protein were infected with VSV G-pseudotyped N- or B-MLV virus-like particles for 4 h and analyzed using the fate-of-capsid assay, as described in Materials and Methods. Total input, supernatants, and pellets were Western blotted using an antibody directed against the p30 capsid protein. “Mouse” refers to the mouse 9230105E10 protein.

Time course of TRIM5αhu-induced loss of particulate N-MLV capsids in infected cells. MDTF cells transduced with either an empty LPCX vector or an LPCX vector expressing TRIM5αhu were incubated at 4°C for 30 min with either N-MLV (A) or B-MLV (B) virus-like particles. The cells were then incubated at 37°C for the indicated times and analyzed using the fate-of-capsid assay. Total input, supernatants, and pellets were Western blotted using an antibody directed against the p30 capsid protein, as described in Materials and Methods.

Influence of MLV capsid residue 110 on the TRIM5αhu-mediated loss of particulate N-MLV capsids. NIH 3T3 and MDTF cells transduced either with an empty LPCX vector or with a vector expressing TRIM5αhu were infected with N-, B-, NBNN-, or BNBB-MLV virus-like particles for 4 h and analyzed using the fate-of-capsid assay, as described in Materials and Methods. Total input, supernatants, and pellets were Western blotted using an antibody directed against the p30 capsid protein. The amount of pelleted p30 was quantified and normalized against total input p30. The percentage of particulate p30 in TRIM5αhu-expressing cells compared to that in the control LPCX cells is provided at the bottom of the figure for each virus.

Effects of deletion of the TRIM5αhu domains on N-MLV restriction and levels of particulate N-MLV capsids in the cytosol of infected cells. (A) MDTF cells transduced with LPCX vectors expressing wild-type TRIM5αhu or deletion mutants were infected with various doses of N-MLV-GFP or B-MLV-GFP. GFP-positive cells were quantified using FACS analysis. (B) Steady-state expression levels of TRIM5 variants are shown. Lysates from MDTF cells transduced with either an empty LPCX vector or LPCX vectors expressing the indicated HA-tagged TRIM5αhu variants were subjected to Western blotting using an anti-HA antibody. The lysates were also blotted against β-actin to control for total protein. (C) MDTF cells transduced with either an empty LPCX vector or vectors expressing the indicated TRIM5αhu variants were infected with VSV G-pseudotyped (Env+) N-MLV virus-like particles for 4 h and analyzed using the fate-of-capsid assay. Cells transduced with the empty LPCX vector were also incubated with N-MLV virus-like particles lacking envelope glycoproteins (Env−). Total input, supernatants, and pellets were Western blotted using an antibody directed against the MLV p30 capsid protein.

Effect of a single amino acid change in the TRIM5αhu B-box 2 domain on N-MLV restriction and the loss of particulate N-MLV capsids. (A) Steady-state expression levels of TRIM5 variants are shown. Lysates from MDTF cells transduced with either an empty LPCX vector or LPCX vectors expressing HA-tagged TRIM5αhu or TRIM5αhu(C95A) were subjected to Western blotting using an anti-HA antibody. The lysates were also blotted against β-actin to control for total protein. (B) MDTF cells transduced with either an empty LPCX vector or LPCX vectors expressing TRIM5αhu or TRIM5αhu(C95A) were infected with various doses of N-MLV-GFP or B-MLV-GFP. GFP-positive cells were quantified using FACS analysis. (C) MDTF cells expressing either an empty LPCX vector or LPCX vectors expressing TRIM5αhu or TRIM5αhu(C95A) were infected with VSV G-pseudotyped (Env+) N-MLV virus-like particles for 4 h and analyzed using the fate-of-capsid assay. The control cells transduced with the empty LPCX vector were also incubated with N-MLV virus-like particles without envelope glycoproteins (Env−). Total input, supernatants, and pellets were Western blotted using an antibody directed against the p30 capsid protein.

Effect of Fv-1 expression on the amount of particulate MLV capsids recovered in the fate-of-capsid assay. (A) MDTF cells transduced with LPCX vectors expressing either Fv-1n or Fv-1b were infected with various doses of N-MLV-GFP or B-MLV-GFP. GFP-positive cells were quantified by FACS analysis. The experiment was repeated with comparable results; the data from a single experiment are shown. (B) MDTF cells expressing either an empty LPCX vector or LPCX vectors expressing TRIM5αhu, Fv-1n, or Fv-1b were incubated with VSV G-pseudotyped N-MLV or B-MLV virus-like particles for 4 h. The virus-containing medium was then replaced with fresh medium, and the cells were lysed 16 h later. Cell lysates were analyzed using the fate-of-capsid assay. Total input, supernatants, and pellets were Western blotted using an antibody directed against the p30 capsid protein.